Production of a living human skin equivalent (LSE) that is economical, mechanically durable and provides effective barrier function for the treatment of epithelial trauma (e.g., burns and diabetic lesions) is a highly prized goal of current efforts in tissue engineering. Stratatech, L.L.C., is currently commercializing various products based on the proprietary human keratinocyte cell line, NIKStm, which has been shown to differentiate into a fully stratified living skin equivalent with normal expression of histological markers. The limited shelf life and high storage costs for currently available skin equivalent products limit their therapeutic potential. We hypothesize that a non-toxic cryoprotectant system based on saccharides specifically designed to form a glass on cooling (i.e., vitrify) rather than freeze will be more effective in the preservation of skin tissue architecture and viability than current methods (i.e., treatment with glycerol). Development of highly effective cryopreservation methods for the NIKStm cell line represents an opportunity to provide the medical community with a much needed supply of off-the-shelf skin graft material with long shelf life at relatively high storage temperatures. PROPOSED COMMERCIAL APPLICATION: As the field of tissue engineering advances, the need for effective, non-toxic preservation methods for future products grows. Stratatech is developing living human skin equivalent products based on our proprietary NIKS (TM) cell line. The goal of this proposal is to develop technology based on recent advances in cryopreservation coupled with methods to introduce protectants into target cells. We hypothesize that by enhancing glass formation a non-toxic, high storage temperature preservation system can be achieved for living human skin equivalents, a model system with high therapeutic value. It is our belief that this technology will be applicable to a wide range of other tissues and organs.